Amyloid particles facilitate surface-catalyzed cross-seeding by acting as promiscuous nanoparticles.

Amyloid seeds are nanometer-sized protein particles that accelerate amyloid assembly as well as propagate and transmit the amyloid protein conformation associated with a wide range of protein misfolding diseases. However, seeded amyloid growth through templated elongation at fibril ends cannot explain the full range of molecular behaviors observed during cross-seeded formation of amyloid by heterologous seeds. Here, we demonstrate that amyloid seeds can accelerate amyloid formation via a surface catalysis mechanism without propagating the specific amyloid conformation associated with the seeds. This type of seeding mechanism is demonstrated through quantitative characterization of the cross-seeded assembly reactions involving two nonhomologous and unrelated proteins: the human Aß42 peptide and the yeast prion-forming protein Sup35NM. Our results demonstrate experimental approaches to differentiate seeding by templated elongation from nontemplated amyloid seeding and rationalize the molecular mechanism of the cross-seeding phenomenon as a manifestation of the aberrant surface activities presented by amyloid seeds as nanoparticles.

The dynamics of the CHO host cell protein profile during clarification and protein A capture in a platform antibody purification process.

Recombinant protein products such as monoclonal antibodies (mAbs) for use in the clinic must be clear of host cell impurities such as host cell protein (HCP), DNA/RNA, and high molecular weight immunogenic aggregates. Despite the need to remove and monitor HCPs, the nature, and fate of these during downstream processing (DSP) remains poorly characterized. We have applied a proteomic approach to investigate the dynamics and fate of HCPs in the supernatant of a mAb producing cell line during early DSP including centrifugation, depth filtration, and protein A capture chromatography. The primary clarification technique selected was shown to influence the HCP profile that entered subsequent downstream steps. MabSelect protein A chromatography removed the majority of contaminating proteins, however using 2D-PAGE we could visualize not only the antibody species in the eluate (heavy and light chain) but also contaminant HCPs. These data showed that the choice of secondary clarification impacts upon the HCP profile post-protein A chromatography as differences arose in both the presence and abundance of specific HCPs when depth filters were compared. A number of intracellularly located HCPs were identified in protein A elution fractions from a Null cell line culture supernatant including the chaperone Bip/GRP78, heat shock proteins, and the enzyme enolase. We demonstrate that the selection of early DSP steps influences the resulting HCP profile and that 2D-PAGE can be used for monitoring and identification of HCPs post-protein A chromatography. This approach could be used to screen cell lines or hosts to select those with reduced HCP profiles, or to identify HCPs that are problematic and difficult to remove so that cell-engineering approaches can be applied to reduced, or eliminate, such HCPs.

Methionine oxidation of Sup35 protein induces formation of the [PSI+] prion in a yeast peroxiredoxin mutant.

The frequency with which the yeast [PSI(+)] prion form of Sup35 arises de novo is controlled by a number of genetic and environmental factors. We have previously shown that in cells lacking the antioxidant peroxiredoxin proteins Tsa1 and Tsa2, the frequency of de novo formation of [PSI(+)] is greatly elevated. We show here that Tsa1/Tsa2 also function to suppress the formation of the [PIN(+)] prion form of Rnq1. However, although oxidative stress increases the de novo formation of both [PIN(+)] and [PSI(+)], it does not overcome the requirement of cells being [PIN(+)] to form the [PSI(+)] prion. We use an anti-methionine sulfoxide antibody to show that methionine oxidation is elevated in Sup35 during oxidative stress conditions. Abrogating Sup35 methionine oxidation by overexpressing methionine sulfoxide reductase (MSRA) prevents [PSI(+)] formation, indicating that Sup35 oxidation may underlie the switch from a soluble to an aggregated form of Sup35. In contrast, we were unable to detect methionine oxidation of Rnq1, and MSRA overexpression did not affect [PIN(+)] formation in a tsa1 tsa2 mutant. The molecular basis of how yeast and mammalian prions form infectious amyloid-like structures de novo is poorly understood. Our data suggest a causal link between Sup35 protein oxidation and de novo [PSI(+)] prion formation.

The Apc5 subunit of the anaphase-promoting complex/cyclosome interacts with poly(A) binding protein and represses internal ribosome entry site-mediated translation.

The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit ubiquitin ligase that mediates the proteolysis of cell cycle proteins in mitosis and G(1). We used a yeast three-hybrid screen to identify proteins that interact with the internal ribosome entry site (IRES) of platelet-derived growth factor 2 mRNA. Surprisingly, this screen identified Apc5, although it does not harbor a classical RNA binding domain. We found that Apc5 binds the poly(A) binding protein (PABP), which directly binds the IRES element. PABP was found to enhance IRES-mediated translation, whereas Apc5 overexpression counteracted this effect. In addition to its association with the APC/C complex, Apc5 binds much heavier complexes and cosediments with the ribosomal fraction. In contrast to Apc3, which is associated only with the APC/C and remains intact during differentiation, Apc5 is degraded upon megakaryocytic differentiation in correlation with IRES activation. Expression of Apc5 in differentiated cells abolished IRES activation. This is the first report implying an additional role for an APC/C subunit, apart from its being part of the APC/C complex.

The physical dimensions of amyloid aggregates control their infective potential as prion particles.

Transmissible amyloid particles called prions are associated with infectious prion diseases in mammals and inherited phenotypes in yeast. All amyloid aggregates can give rise to potentially infectious seeds that accelerate their growth. Why some amyloid seeds are highly infectious prion particles while others are less infectious or even inert, is currently not understood. To address this question, we analyzed the suprastructure and dimensions of synthetic amyloid fibrils assembled from the yeast (Saccharomyces cerevisiae) prion protein Sup35NM. We then quantified the ability of these particles to induce the [PSI+] prion phenotype in cells. Our results show a striking relationship between the length distribution of the amyloid fibrils and their ability to induce the heritable [PSI+] prion phenotype. Using a simple particle size threshold model to describe transfection activity, we explain how dimensions of amyloid fibrils are able to modulate their infectious potential as prions.

Interaction of hnRNP-C1/C2 proteins with RNA: analysis using the yeast three-hybrid system.

Three-hybrid assays for the analysis of RNA-protein interactions in vivo are usually used, due to technical limitations, only for RNA baits that do not contain runs of four or more consecutive uridines. The present study provides the first example of a three-hybrid analysis of synthetic and natural uridine-rich RNA sequences. The use of the three-hybrid assay enabled us to demonstrate a functional difference between two closely related proteins, heterogeneous nuclear ribonucleoprotein C1 (hnRNP-C1) and hnRNP-C2. The hnRNP-C2 protein, an alternatively spliced variant of hnRNP-C1, contains an additional 13 amino acids between an RNA binding domain (RBD) and a basic leucine zipper-like motif (bZLM), also implied in RNA binding. This study shows that (i) for efficient binding of hnRNP-C1/C2 to RNA, the context of the U-stretch is more important than the stretch itself; (ii) both the RBD and the bZLM bind RNA independently; and (iii) the C2-related 13-amino acid insert enhances the specificity of either the RBD, the bZLM, or the full-length protein towards its ligand, allowing it to bind only the most high-affinity sequences while discriminating against those that do not perfectly match this category. The three-hybrid system is a powerful tool to work out the functional significance of peptide 'modules' within RNA binding proteins generated by alternative splicing.

Cellular factors important for the de novo formation of yeast prions.

Prions represent an unusual structural form of a protein that is 'infectious'. In mammals, prions are associated with fatal neurodegenerative diseases such as CJD (Creutzfeldt-Jakob disease), while in fungi they act as novel epigenetic regulators of phenotype. Even though most of the human prion diseases arise spontaneously, we still know remarkably little about how infectious prions form de novo. The [PSI+] prion of the yeast Saccharomyces cerevisiae provides a highly tractable model in which to explore the underlying mechanism of de novo prion formation, in particular identifying key cis- and trans-acting factors. Most significantly, the de novo formation of [PSI+] requires the presence of a second prion called [PIN+], which is typically the prion form of Rnq1p, a protein rich in glutamine and aspartic acid residues. The molecular mechanism by which the [PIN(+)] prion facilitates de novo [PSI+] formation is not fully established, but most probably involves some form of cross-seeding. A number of other cellular factors, in particular chaperones of the Hsp70 (heat-shock protein 70) family, are known to modify the frequency of de novo prion formation in yeast.

Effect of Ku proteins on IRES-mediated translation.

BACKGROUND INFORMATION: Ku is an abundant nuclear heterodimeric protein composed of 70 and 86 kDa subunits. As an activator of the catalytic subunit of DNA-PK (DNA-dependent protein kinase), Ku plays an important role in DNA repair and recombination. Ku is also involved in actions independent of DNA-PK, such as transcription regulation and telomere maintenance. Although Ku is localized in the cytoplasm under specific cellular conditions, no functions for Ku outside of the nucleus have as yet been reported. In addition to DNA binding, Ku binds specific RNA sequences with high affinity. However, no specific cellular mRNA targets for Ku have been identified. RESULTS: In a yeast three-hybrid system, Ku70 bound to an RNA bait that contained an IRES (internal ribosomal entry site) element. A single band with migration properties similar to those of Ku70 was immunoprecipitated with anti-Ku antibody, using UV cross-linked complexes formed by HeLa cell nuclear extracts and an IRES-containing RNA probe. IRES activity was reduced in Ku80(-/-) cells. Overexpression of Ku proteins stimulated IRES-dependent translation. CONCLUSIONS: The present study suggests that Ku binds IRES elements within RNA molecules, and that Ku plays a role in the modulation of IRES-mediated mRNA translation.